It is generally accepted that N-linked glycosylation in the IgG CH2 domain is required for functional engagement of activating FcγR receptors. For example, Jefferis et al., reported that “[o]ne of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by FcRs although the carbohydrate moieties are on the periphery of the FcRIII-Fc interface” (Jefferis at al., J. Biol. Chem. 276:45539, 2001). Similarly, other researchers have stated, “[g]lycosylation of IgG at the conserved asparagine residue at position 297, which is in the CH2 domain (immunoglobulin heavy-chain constant domain 2), is required to support the interaction between IgG and FcγRs, which is a prerequisite for ADCC80” (Nat. Rev. Immunol. 6:343, 2006).
The need for specific N-linked glycosylation necessitates the use of mammalian cell cultures for antibody production. However, the cost for antibodies manufactured in that way is considerable, and it is difficult and expensive to build sufficient capacity for the rising number of antibody drugs in development. Antibodies are among the most expensive of all drugs where the annual cost per patient can be 35,000 USD or more. The high cost reflects the fact that antibodies are now marketed for chronic conditions and of their relatively low potency results in the need for high cumulative doses. Consequently, expensive large-scale production capacity is currently required to fulfill market demand and produce tens to hundreds of kilograms of product per year. Use of bacterial fermentation to produce IgG would be economically beneficial. A process that routinely takes several months in mammalian systems can take as little as one month with E. coli. Fermentation capital costs for therapeutic proteins are also lower for bacterial production systems (J. Immunol. Meth. 263:133, 2002). Unfortunately, when wild type IgGs are produced in E. coli, they do not bind Fc receptors. E. coli-produced aglycosylated IgG1 failed to bind C1q and the FcγRI receptor, suggesting to prior investigators that the E. coli-derived IgG1 lacked effector functions (J. Immunol. Meth. 263:133, 2002).
Effector functions are a key component of efficacy for anti-cancer antibodies, one of the primary targets for antibody drug development. Antibody activity in vivo requires efficient interaction between the antibody and cellular Fc-receptors on innate immune effector cells, and it has been reported that the cytotoxic activity of antibodies in vivo is mainly determined by the co-engagement of activating and inhibitory FcRs (Curr. Op. Immunol. 19:239, 2007). In fact, in the absence of functional Fc/FcR interactions, several therapeutic antibodies were completely ineffective in mouse xenografted tumor models (Nature Med. 6:433, 2000).